Abstract
Of 12 naturally occurring, activating mutations in the seven-transmembrane (7TM) domain of the human Ca2+ receptor (CaR) identified previously in subjects with autosomal dominant hypocalcemia (ADH), five appear at the junction of TM helices 6 and 7 between residue Ile819 and Glu837. After identifying a sixth activating mutation in this region, V836L, in an ADH patient, we studied the remaining residues in this region to determine whether they are potential sites for activating mutations. Alanine-scanning mutagenesis revealed five additional residues in this region that when substituted by alanine led to CaR activation. We also found that, whereas E837A did not activate the receptor, E837D and E837K mutations did. Thus, region Ile819-Glu837 of the 7TM domain represents a "hot spot" for naturally occurring, activating mutations of the receptor, and most of the residues in this region apparently maintain the 7TM domain in its inactive configuration. Unique among the residues in this region, Pro823, which is highly conserved in family 3 of the G protein-coupled receptors, when mutated to either alanine or glycine, despite good expression severely impaired CaR activation by Ca2+. Both the P823A mutation and NPS 2143, a negative allosteric modulator that acts on the 7TM through a critical interaction with Glu837, blocked activation of the CaR by various ADH mutations. These results suggest that the 7TM domain region Ile819-Glu837 plays a key role in CaR activation by Ca2+. The implications of our finding that NPS 2143 corrects the molecular defect of ADH mutations for treatment of this disease are also discussed.
Highlights
Of 12 naturally occurring, activating mutations in the seven-transmembrane (7TM) domain of the human Ca2؉ receptor (CaR) identified previously in subjects with autosomal dominant hypocalcemia (ADH), five appear at the junction of TM helices 6 and 7 between residue Ile819 and Glu837
Expression and Characterization of Naturally Occurring, Activating CaR Mutations Identified in the TM6/TM7 Junction Region—We identified a novel heterozygous germline, missense mutation V836L, in a subject with ADH.2
To assess the functional significance of this and five previously reported ADH mutations located in this region of the CaR (12–16), we measured PI hydrolysis as a function of extracellular Ca2ϩ concentration and CaR expression by immunoblot in HEK-293 cells transfected with WT and mutant CaR cDNAs
Summary
CaR, extracellular Ca2ϩ receptor; GPCR, G protein-coupled receptor; VFT, Venus’s flytrap; 7TM, seventransmembrane domain; ADH, autosomal dominant hypocalcemia; hCaR, human extracellular Ca2ϩ receptor; DMEM, Dulbecco’s modified Eagle’s medium; PI, phosphoinositide; PIPES, 1,4-piperazinediethanesulfonic acid; [Ca2ϩ]o, extracellular calcium ion; WT, wild type. Agonist binding to the cleft of the VFT leads to VFT closure and a 70° rotation of one monomer relative to the other about an axis perpendicular to the dimer interface How this agonist-induced conformational change in the VFT domain is transmitted to the 7TM domain to cause CaR activation is a key unanswered question. Ten ADH mutations cluster between residues 116 –131 in a part of the VFT domain, loop 2, which is the site of intermolecular disulfide-linked dimerization (6) The corresponding loop in the glutamate type 1 receptor three-dimensional structure represents a “switch region” in that it is disordered in the active, agonist-bound form of the VFT, but in the inactive form residues equivalent to 117–123 of the CaR become ␣-helical. We identified multiple additional TM6/TM7 residues that lead to receptor activation upon substitution of alanine and a unique mutation in TM6, P823A, that blocks CaR activation, mimicking the effect of a negative allosteric modulator
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